Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that plays key roles in brain development, neuronal survival, and nervous system remodeling. Formation of SIP in neurons by sphingosine kinases (SphKs) is stimulated by a variety of agonists, including nerve growth factor (NGF) and neurotrophin-3 (NT- 3), and many of its functions are mediated by its cell surface receptors (S1P1-5). However, there is accumulating evidence suggesting that S1P also has receptor-independent intracellular effects, particularly those related to neuronal survival. A key factor hindering understanding of the intracellular roles of S1P is the lack of identification of bona fide intracellular targets. Cellular localizations of the two SphK isoenzymes that produce S1P, SphK1 and SphK2, have begun to provide some clues. SphK1 is mainly cytosolic and is activated and translocated to the plasma membrane to the vicinity of its receptors by many stimuli. In contrast, in many cell types, SphK2 is mainly found in the nucleus where its function is not known. My preliminary data indicate that S1P does bind to at least one protein that is present in the nucleus, and that it can also alter the DMA binding activity of several transcription factors. The main hypothesis of this proposal is that S1P interacts with specific intracellular targets that mediate its intracellular and/or intranuclear actions. S1P produced in the nucleus may modulate gene expression by binding to a transcription factor or to some other regulator, and the objective of this proposal is the elucidation of the direct intracellular targets of S1 P. To this end, I will identify intracellular proteins that bind S1P and investigate the effect of S1P on transcription factor DNA binding activity using a transcription factor activity membrane that allows simultaneous activity profiling of multiple transcription factors. In addition, I will determine whether intracellularly generated S1P by SphK1 or SphK2 modulates distinct transcription factor activities. Finally, the significance of the specific cellular location of S1P will be determined by altering the expression of the two sphingosine kinase isoenzymes, which are localized in different subcellular compartments. This will be accomplished by downregulation of expression of each of the kinases with small interfering RNA, and observing the effects on transcription factor activity and gene expression. Intracellular S1P enhances neuronal survival and suppresses cell death, key processes involved in neurodegenerative disorders such as stroke and Alzheimer's and Parkinson's diseases. Deciphering the mechanism of action of this sphingolipid metabolite could provide clues for the prevention and treatment of devastating human neurodegenerative diseases.